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Articles 1 - 30 of 80
Full-Text Articles in Life Sciences
Elucidating The Priming Mechanism Of Clpxp Protease By Single-Domain Response Regulator Cpdr In Caulobacter Crescentus, Kimberly E. Barker
Elucidating The Priming Mechanism Of Clpxp Protease By Single-Domain Response Regulator Cpdr In Caulobacter Crescentus, Kimberly E. Barker
Masters Theses
In Caulobacter crescentus, progression through the cell cycle is regulated by the AAA+ protease ClpXP, and there are several classes of cell-cycle substrates that require adaptors in order to be degraded. CpdR, a single domain-response regulator, binds the N-terminal domain of ClpXP and primes the protease for degradation of downstream factors (Lau et al., 2015). The ability of CpdR to bind ClpX is regulated by its phosphorylation state. In the unphosphorylated state, CpdR binds ClpXP and guides its localization to the cell pole during the swarmer to stalked transition, where CpdR is mediates degradation of substrates such as PdeA. …
Beyond The Fat: Protein Metabolism And Muscle Function As Part Of The Migratory Syndrome Of Songbirds, Cory R. Elowe
Beyond The Fat: Protein Metabolism And Muscle Function As Part Of The Migratory Syndrome Of Songbirds, Cory R. Elowe
Doctoral Dissertations
Migration allows animals to seasonally exploit favorable habitats that are geographically disparate, and migratory animals have a suite of adaptations to complete their extraordinary journeys. This “migratory syndrome” of traits is comprised of seasonally-responsive adaptations that together result in changes to metabolism, body composition, and reproductive status, all of which act to increase the probability of a successful migration. The focus of my dissertation is to investigate the suite of changes in metabolism and muscle function that enable birds to undertake long-duration flights. In chapter 1, I compare fuel use in flight between two related migratory warbler species that migrate …
The Genomic Dynamics Of Fermented Food Microbes, Katherine Chacón-Vargas
The Genomic Dynamics Of Fermented Food Microbes, Katherine Chacón-Vargas
Doctoral Dissertations
Along with the agriculture and domestication revolution, humans have utilized bacteria, yeasts, and molds for millennia in the production of traditionally fermented foods and beverages. Fermentation is a very ancient practice of high relevance nowadays since it contributes with a great variety of foods worldwide. Microbial fermentation allows metabolic transformation of the raw food materials leading to biochemical changes that played a key role in food preservation, health benefits, nutrition, flavors, and texture, among others. Food fermentation practices could diverge from traditional artisanal spontaneous fermentation to industrialize methods with specialized microbial starters and although fermented environments tend to be very …
Comparison Of The Humoral Immune Response Following Both Bacterial Challenge And Rnai Of Major Factors On Proliferation Of Bartonella Quintana In The Human Louse, Jake Zina
Masters Theses
Human body lice, Pediculus humanus humanus, and head lice, Pediculus humanus capitis, have been hematophagous ectoparasites of humans for thousands of years. Despite being ecotypes, only body lice are known to transmit bacterial diseases to humans, and it appears that lower humoral and cellular immune responses allow body lice to possess a higher vector competence. We previously observed that the transcription level of the defensin 1 gene was up-regulated only in head lice following oral challenge of Bartonella quintana, a causative agent of trench fever, and also that body lice excreted more viable B. quintana in their …
Changes In Gene Expression From Long-Term Warming Revealed Using Metatranscriptome Mapping To Fac-Sorted Bacteria, Christopher A. Colvin
Changes In Gene Expression From Long-Term Warming Revealed Using Metatranscriptome Mapping To Fac-Sorted Bacteria, Christopher A. Colvin
Masters Theses
Soil microbiomes play pivotal roles to the health of the environment by maintaining metabolic cycles. One question is how will climate change affect soil bacteria over time and what could the repercussions be. To answer these questions, the Harvard Forest Long-Term Warming Experiment was established to mimic predicted climate change by warming plots of land 5℃ above ambient conditions. In 2017, 14 soil core samples were collected from Barre Woods warming experiment to mark 15 years since the establishment of the soil warming in that location. These samples underwent traditional metatranscriptomics to generate an mRNA library as well as a …
Chemosensory Receptors In Berghia Stephanieae: Bioinformatics And Localization, Kelsi L. Watkins
Chemosensory Receptors In Berghia Stephanieae: Bioinformatics And Localization, Kelsi L. Watkins
Masters Theses
Chemosensation is achieved through the binding of chemical signals to chemoreceptor proteins embedded in the membranes of sensory neurons. The molecular identity of these receptors, as well as the downstream processing of chemosensory signals, has been well studied in arthropods and vertebrates. However, very little is known about molluscan chemosensation. The identity of chemoreceptor proteins in the nudibranch mollusc Berghia stephanieae are unknown. Data from other protostome and molluscan studies suggest Berghia may use ionotropic receptors for some forms of chemoreception. This study used a bioinformatics approach to identify potential chemosensory ionotropic receptors in the transcriptome of Berghia. A …
Flexible Phenotypes, Energetics, And Whole-Animal Performance Of Migratory Songbirds, Michael Griego
Flexible Phenotypes, Energetics, And Whole-Animal Performance Of Migratory Songbirds, Michael Griego
Doctoral Dissertations
Animal life has evolved innumerable strategies to adapt to a great range of environmental conditions present on earth. The physiology of free-living animals has thus been shaped to allow for maximal performance under challenging conditions and has given rise to traits that enable animals to overcome daunting ecological pressures. Few life history stages in the animal kingdom rival the intensity of annual avian migration: the extreme metabolic requirements of long-distance flight coupled with navigating vast and hostile ecological barriers results in enormously high mortality for young birds. It is therefore the main focus of this thesis to identify physiological traits …
Constraints Of The Imagination: How Phenotypes Are Shaped Through Genetics, The Environment, And Development, Michelle Gilbert
Constraints Of The Imagination: How Phenotypes Are Shaped Through Genetics, The Environment, And Development, Michelle Gilbert
Doctoral Dissertations
Phenotypic constraints are ubiquitous throughout nature, being found throughout all stages of life and at multiple different biological levels including cellular, genetic, environmental, behavioral, evolutionary, and developmental. These constraints have shaped, not only the natural world, but the way that we perceive what is possible, or impossible, an observation made clear by François Jacob in his 1977 paper “Evolution and Tinkering”. This is reflected in the literature, repeatedly, by the regular occurrence of densely packed visualization of phenotypic space that seemingly always have large areas that go unoccupied. Despite constrained regions of space being observable across countless taxa, identifying the …
Tracking And Modulating Circadian Rhythms In Cell Culture Models, Sujeewa Sampath Lellupitiyage Don
Tracking And Modulating Circadian Rhythms In Cell Culture Models, Sujeewa Sampath Lellupitiyage Don
Doctoral Dissertations
Circadian rhythms are 24–hour activity cycles that exist for all organisms, from yeast and bacteria to mammals. Epidemiological data has shown that disruptions to circadian rhythms are associated with various diseases, including cancers. Circadian rhythms are regulated at the cellular level by a "molecular clock,” whose oscillations in protein transcription and translation control a range of downstream pathways. However, the connections between altered rhythms and diseases at the molecular level is unclear. Therefore, in this project, I tracked circadian rhythms in a high-resolution manner and thoroughly analyzed them to more completely reflect circadian behaviors and clock functioning at the molecular …
Screening For Binding Partners And Protein-Protein Interactions Of A Fungal Transcription Factor- Xdr1, Nishadi Punsara Gallala Gamage
Screening For Binding Partners And Protein-Protein Interactions Of A Fungal Transcription Factor- Xdr1, Nishadi Punsara Gallala Gamage
Masters Theses
Clarireedia spp. (formerly Sclerotinia homoeocarpaF.T. Bennett) is the causal agent dollar spot, the most economically important turfgrass disease impacting golf courses in North America. The most effective strategy for dollar spot control is repeated application of multiple classes of fungicides. However, reliance on chemical application has led to resistance to four classes of fungicides as well as multidrug resistance (MDR). Fungi are known to detoxify xenobiotics, like fungicides, through transcriptional regulation of three detoxification phases: modification, conjugation and secretion. Little is known, however, of the protein-protein interactions that facilitate these pathways. Following next-generation RNA sequencing of Clarireedia spp., a …
Drivers And Direct Impacts Of Lean Mass Dynamics On The Stopover Ecology And Migratory Pace Of Nearctic-Neotropical Migrant Songbirds In Spring, Mariamar Gutierrez Ramirez
Drivers And Direct Impacts Of Lean Mass Dynamics On The Stopover Ecology And Migratory Pace Of Nearctic-Neotropical Migrant Songbirds In Spring, Mariamar Gutierrez Ramirez
Doctoral Dissertations
Annual migration in songbirds is one of the most demanding life-history stages. It represents a period of high mortality, yet there is still much unknown about the ecological correlates that influence its successful completion. After long non-stop migratory flights, birds require a stopover period to rest and replenish depleted energy reserves. Birds use fat as the primary fuel to power long-distance flights. However, birds also burn lean tissue, which results in significant reductions in muscle and organ masses. The discovery and quantification of lean mass catabolism represented a paradigm shift in migration ecology because non-fat components were thought to remain …
A Tipping Point In The Ecuadorian Amazon Rainforest: Current And Future Land-Use And Climate Change Trends, Alula Shields
A Tipping Point In The Ecuadorian Amazon Rainforest: Current And Future Land-Use And Climate Change Trends, Alula Shields
Masters Theses
Many regions of the Amazon are experiencing drastic changes as deforestation and climate change drive the world’s largest continuous rainforest towards a ‘tipping point’. These disturbances are changing natural cycles that once past a critical threshold, will mark an unstoppable transition to an altered ecosystem. Losing areas of the Amazon rainforest will have implications for the global climate, global carbon budget, and global hydrological regimes. Scholars have projected these tipping points for areas of the eastern Amazon rainforest, but much less scholarship focuses on the headwaters of the Western Amazon, an area of great cultural and biological importance. Ecuador is …
Functional Morphology, Biomechanics, And Evolution Of Ruminant Mammals, Abby Vander Linden
Functional Morphology, Biomechanics, And Evolution Of Ruminant Mammals, Abby Vander Linden
Doctoral Dissertations
Ruminant mammals, including the families Bovidae, Cervidae, Tragulidae, Moschidae, Antilocapridae, and Giraffidae, display incredible past and present diversity in morphology, ecology, and behavior. They inhabit an impressive range of environments across North and South America, Europe, Asia, and Africa, and compel the fascination of naturalists and researchers alike with their charismatic social behavior and conspicuous cranial appendages. I explore the drivers and consequences of this spectacular diversity through a comparative morphological framework, biomechanical modeling approaches, and semi-parametric and likelihood-based methods for estimating state-dependent diversification rates across the ruminant phylogeny. Together, these investigations provide evidence for adaptation via correlated evolution of …
The Time Course And Neuroanatomy Of Rhinophore Regeneration In The Nudibranch Berghia Stephanieae, Ani Maroyan
The Time Course And Neuroanatomy Of Rhinophore Regeneration In The Nudibranch Berghia Stephanieae, Ani Maroyan
Masters Theses
Within five weeks, the nudibranch, Berghia stephanieae (Gastropoda, Mollusca) can regenerate a severed rhinophore, the main olfactory appendage, such that it is indistinguishable from a non-lesioned rhinophore. The rhinophore is a 2 mm long stalk with lateral sides covered in ridges and the distal third covered in fluorescent pigmentation. Its internal morphology is dominated by longitudinal musculature, overlaid by a complex neuronal plexus, which contains neurons that express various neuroactive substances including serotonin and small cardioactive peptide (SCP). Two large nerves originate in the rhinophore ganglion at the base of the rhinophore and run the length of the rhinophore. To …
Development Of A New Behavioral Assay For Juvenile Berghia Stephanieae, Kelly E. Fischer
Development Of A New Behavioral Assay For Juvenile Berghia Stephanieae, Kelly E. Fischer
Masters Theses
Developing robust behavioral assays to study olfactory-driven behaviors allows for greater insight into the neural mechanisms behind them. Oftentimes, olfactory behavioral assays require a two-choice design, consistent variables, and controlled stimulus application. This can be challenging when working with marine dwelling organisms such as nudibranchs. Extensive work shows the importance of olfaction in both pre-metamorphic larval development and adult stage nudibranchs (Gastropoda, Mollusca). However, there is little research investigating how olfaction plays a role in rapidly developing, post-metamorphic juvenile nudibranchs such as Berghia stephanieae. To study olfactory-associated behaviors in juvenile Berghia, a novel behavioral microfluidic chip was designed which …
Monitoring Mammals At Multiple Scales: Case Studies From Carnivore Communities, Kadambari Devarajan
Monitoring Mammals At Multiple Scales: Case Studies From Carnivore Communities, Kadambari Devarajan
Doctoral Dissertations
Carnivores are distributed widely and threatened by habitat loss, poaching, climate change, and disease. They are considered integral to ecosystem function through their direct and indirect interactions with species at different trophic levels. Given the importance of carnivores, it is of high conservation priority to understand the processes driving carnivore assemblages in different systems. It is thus essential to determine the abiotic and biotic drivers of carnivore community composition at different spatial scales and address the following questions: (i) What factors influence carnivore community composition and diversity? (ii) How do the factors influencing carnivore communities vary across spatial and temporal …
Diversity And Evolution Of Human Eccrine Sweat Gland Density, Andrew W. Best
Diversity And Evolution Of Human Eccrine Sweat Gland Density, Andrew W. Best
Doctoral Dissertations
Human eccrine density is highly derived. However, little is known about contemporary variation in this trait, what shapes it, and how it influences heat dissipation. This project explores 3 questions: 1) Is variation in functional eccrine density (FED) explained by childhood climate? 2) Is this variation patterned by geographic ancestry? 3) Is variation in FED associated with differences in heat dissipation capacity? We measured FED and sweat production in 6 body areas via pharmacological stimulation and impressions of sweating skin in 72 participants. Childhood climate variables were taken from the WorldClim database and geographic ancestry was estimated with 23andMe tests. …
Mechanisms Of Mitotic Checkpoint Silencing By The Disordered Kinetochore Protein Spc105, Margaux Audett
Mechanisms Of Mitotic Checkpoint Silencing By The Disordered Kinetochore Protein Spc105, Margaux Audett
Doctoral Dissertations
The kinetochore protein Spc105R (DmSpc105R) is a large intrinsically disordered protein (IDP) that recruits spindle assembly checkpoint (SAC) proteins and is required for SAC signaling in a conserved manner. Chromosome biorientation satisfies the SAC and while it has been proposed that SAC satisfaction may require the establishment of stable kinetochore-MT (KT-MT) attachments and tension generation, the question of whether tension directly regulates SAC signaling is unresolved and controversial. Here we present data in support of Spc105R as a tension-sensing IDP that directly regulates checkpoint protein localization and signaling independent of the establishment of stable KT-MT attachments. The N-terminus of DmSpc105R …
Factors Influencing Primate Hair Microbiome Diversity, Catherine Kitrinos
Factors Influencing Primate Hair Microbiome Diversity, Catherine Kitrinos
Masters Theses
Primate hair is both a substrate upon which essential social interactions occur and an important host-pathogen interface. As commensal microbes provide important immune functions for their hosts, understanding the microbial diversity in primate hair could provide insight into primate immunity and disease transmission. While studies of human hair and skin microbiomes show differences in microbial communities across body regions, little is known about the nonhuman primate hair microbiome. In this study, we collected hair samples (n=159) from 8 body regions across 12 nonhuman primate species housed at 3 US institutions to examine 1) the diversity and composition of the primate …
Dynamic Evolution In The Leucine-Rich Repeat Receptor-Like Kinase (Lrr-Rlk) Family Of Receptors, Jarrett Man
Dynamic Evolution In The Leucine-Rich Repeat Receptor-Like Kinase (Lrr-Rlk) Family Of Receptors, Jarrett Man
Doctoral Dissertations
Cells in multicellular organisms must gauge their environmental conditions, including neighboring cells, during development. In plants, the Leucine-Rich Repeat Receptor-like Kinases (LRR-RLKs) encode a family of membrane-bound receptors that transduce such cell-to-cell signals and are required for many aspects of plant development. Very little is known about the function of most of these genes, and the evolutionary history of the family is difficult to infer because of its size and complexity. Several factors contribute to this difficulty, including genetic redundancy, challenging bioinformatic detection, exceptionally large family size, and high copy number variation among species. In this dissertation, I characterize some …
Engineering Mesothelin-Binding Proteins As Targeted Cancer Diagnostics And Therapeutics, Allison Rita Sirois
Engineering Mesothelin-Binding Proteins As Targeted Cancer Diagnostics And Therapeutics, Allison Rita Sirois
Doctoral Dissertations
Cancer is a significant global health concern; and traditional therapies, including chemotherapeutics, are often simultaneously toxic yet ineffective. There is a critical need to develop targeted cancer therapeutics which specifically inhibit molecules or molecular pathways essential for tumor growth and maintenance. Furthermore, a targeted therapy is only effective when a patient's tumor expresses the molecular target; therefore, companion diagnostics, including molecular imaging agents, are a necessary counterpart of targeted therapies. Mesothelin (MSLN) is a cell surface protein overexpressed in numerous cancers, including triple-negative breast, pancreatic, ovarian, liver, and lung, with limited expression in normal tissues. Aberrant MSLN expression promotes tumor …
Origin Of Gene Specificity In The Nitrogen-Fixing Symbiosis, Christina Marie Stonoha-Arther
Origin Of Gene Specificity In The Nitrogen-Fixing Symbiosis, Christina Marie Stonoha-Arther
Doctoral Dissertations
Many legumes form a symbiosis with nitrogen-fixing bacteria found in the soil. This relationship is beneficial to both the plant and the bacteria; the plant receives nitrogen that is otherwise limited, and the bacteria receive fixed carbon. Upon sensing the bacteria, the plant forms a new organ (the nodule) where the bacteria are housed within the cells. Many genes are required for the proper formation and function of nodules; this dissertation is broadly focused on how genes required for nitrogen-fixing symbiosis are co-opted from other cellular processes and how they are specialized for symbiosis. Protein trafficking from the plant to …
Physiology Of A Basal Vertebrate, The Sea Lamprey (Petromyzon Marinus): Osmoregulation And Corticosteroid Action, Ciaran A. S. Shaughnessy
Physiology Of A Basal Vertebrate, The Sea Lamprey (Petromyzon Marinus): Osmoregulation And Corticosteroid Action, Ciaran A. S. Shaughnessy
Doctoral Dissertations
Lamprey represent the most basal living example of a vertebrate animal which regulates its internal fluid and ion homeostasis. This phylogenetic position among vertebrates makes lamprey an important model organism for understanding the basal state, and thus the evolution, of physiological systems such as the mechanisms of osmo- and ionoregulation and the endocrine factors controlling them. Sea lamprey (Petromyzon marinus) are an anadromous fish, migrating from freshwater (FW) to seawater (SW) as juveniles, then returning back upstream as mature adults to spawn. Surviving this transition from a solute-poor FW environment to a solute-concentrated SW environment requires many changes …
Improved Molecular Diagnostics For Soil-Transmitted Helminths, Nils Pilotte
Improved Molecular Diagnostics For Soil-Transmitted Helminths, Nils Pilotte
Doctoral Dissertations
Current World Health Organization recommendations for the diagnosis of soil-transmitted helminths (STH) rely on antiquated microscopy-based techniques that lack both diagnostic sensitivity and specificity. While sufficient for providing rough estimates of infection frequency and intensity within high prevalence settings, these techniques lack the capacity to effectively estimate infection levels following successful intervention efforts, as worm burdens decline and community prevalences decrease. Furthermore, an expanding body of evidence is suggesting that microscopy-based misdiagnosis of infection is likely a larger concern then previously believed. As such, with an increase in programmatic support for transmission interruption and an escalating belief in the possibility …
Tpr-Containing Proteins Control Protein Organization And Homeostasis For The Endoplasmic Reticulum, Jill Bradley-Graham
Tpr-Containing Proteins Control Protein Organization And Homeostasis For The Endoplasmic Reticulum, Jill Bradley-Graham
Doctoral Dissertations
The endoplasmic reticulum (ER) is a complex, multifunctional organelle comprised of a continuous membrane and lumen that is organized into several functional regions. It plays various roles including protein translocation, folding, quality control, secretion, calcium signaling, and lipid biogenesis. Cellular protein homeostasis is maintained by a complicated chaperone network, and the largest functional family within this network consists of proteins containing tetratricopeptide repeats (TPRs). TPRs are well-studied structural motifs that mediate intermolecular protein-protein interactions, supporting interactions with a wide range of ligands or substrates. Nine TPR-containing proteins have been shown to localize to the ER and control protein organization and …
De-Coding The Impact Of Evolved Changes In Gene Expression And Cellular Phenotype On Primate Evolution, Trisha Zintel
De-Coding The Impact Of Evolved Changes In Gene Expression And Cellular Phenotype On Primate Evolution, Trisha Zintel
Doctoral Dissertations
The goal of the dissertation work outlined here was to investigate the influence of proximal processes contributing to evolutionary differences in phenotypes among primate species. There are numerous previous comparative analyses of gene expression between primate brain regions. However, primate brain tissue samples are relatively rare, and my results have contributed to the pre-existing data on more well-studied primates (i.e. humans, chimpanzees, macaques, marmosets) as well as produced information on more rarely-studied primates (i.e. patas monkey, siamang, spider monkey). Additionally, the primary visual cortex has not previously been as extensively studied at the level of gene expression as other brain …
Improving Growth And Survival Of Cultured Yellow Lampmussel (Lampsilis Cariosa) For Restoring Populations, Virginia Martell
Improving Growth And Survival Of Cultured Yellow Lampmussel (Lampsilis Cariosa) For Restoring Populations, Virginia Martell
Masters Theses
In North America 72% of freshwater mussel species are endangered, threatened, or of special concern due to factors such as habitat loss and degradation, biological invasion, and land use change. Propagation of freshwater mussels has been considered a necessary conservation strategy for population restoration where threats have been mitigated but small population sizes limit population viability. Yellow lampmussel is a species of freshwater mussel that is endangered, threatened, or imperiled throughout its range; therefore, I evaluated laboratory techniques (probiotic supplements and secondary rearing designs) to improve culture of yellow lampmussel for population restoration. Several aquaculture facilities commonly use probiotics; thus, …
Engineering Of An Antibody-Conjugated Nanogel Platform For Targeted Drug Delivery To T Lymphocytes, Mine Canakci
Engineering Of An Antibody-Conjugated Nanogel Platform For Targeted Drug Delivery To T Lymphocytes, Mine Canakci
Doctoral Dissertations
In an ideal chemotherapy, cytotoxic drugs travel through the bloodstream, reach cells all over the body and preferentially kill abnormal cells. Yet, the hydrophilic or lipophilic property of the small-molecule drugs affects their ability to reach cells from the bloodstream. So, only a small portion of the drug reaches to the diseased tissue. A selective cell killing approach for cancer therapy gained momentum after the realization that cancer cells carry unique set of molecular markers on their cell surface. The development of antibody drug conjugates (ADC) revolutionized the targeted approach for drug delivery. ADCs are composed of cytotoxic agents covalently …
Characterizing The Interaction Between Non-Pathogenic Fusarium Oxysporum And Arabidopsis Thaliana To Determine Beneficial Effects Conferred To The Model Plant Host, Kathryn Isabelle Vescio
Characterizing The Interaction Between Non-Pathogenic Fusarium Oxysporum And Arabidopsis Thaliana To Determine Beneficial Effects Conferred To The Model Plant Host, Kathryn Isabelle Vescio
Masters Theses
Fusarium oxysporum (Fo) is a soil-borne fungal pathogen that causes vascular wilt disease on a broad range of plants, including agricultural crops and the model plant Arabidopsis thaliana. There are non-pathogenic members of the Fo species complex that confer defense benefits against other pathogens to the host plant, however alteration to the host’s physiology through interaction with one of these strains, Fo47, have not been described. In this study, we aimed to establish the Fo47-A. thaliana interaction and determine if Fo47 reduces disease severity of a pathogenic Fo isolate, Fo5176. Additionally, we sought to use bioinformatics to mine …
Investigating The Genetic Basis Of And Plasticity In Ecologically Relevant Phenotypes In African Cichlids, Dina Navon
Investigating The Genetic Basis Of And Plasticity In Ecologically Relevant Phenotypes In African Cichlids, Dina Navon
Doctoral Dissertations
Understanding the generation of phenotypic variation by linking it to genetic variation has long been a focus of evolutionary biology; this framework has successfully been implemented in a variety of studies across the tree of life1,2. However, our understanding of the phenotype remains incomplete until we account for a myriad of interactions that influence the genotype-phenotype map, including interactions between traits (TxT), interactions between genes and the environment (GxE), as well as the ways in which various types of interactions are nested within and build upon one another (e.g., (TxT)xG). My dissertation aims to contribute to filling this …